A central goal in Neurobiology has been the discovery of ways in which to rescue dopamine (DA) neurons from the progressive degeneration that occurs during aging or in Parkinson's disease and to replace lost tissue with transplanted cells capable of DAergic function. These strategies depend for their success on a reliable source of transplantable DA neurons and the identification of factors relevant to their growth and survival. Unfortunately, progress on both of these fronts has been greatly impeded by the fact that DA neurons comprise less than 1 percent of the total cells currently found in mesencephalic cultures and transplants. Discovering ways in which to segregate DA neurons from other cell types poses a significant challenge, but a necessary next step. In our original grant, we proposed to improve these ratios by attempting to separate DA neurons from other cells in the brain by flow cytometry following retrograde labeling with fluorescent dyes (diI). Over the last 2.5 years, we have explored a number of alternative ways in which to isolate pure populations of DA neurons from the brain or persuade cells to behave as DA neurons (ie. express the DA biosynthetic enzyme tyrosine hydroxylase; TH). These studies suggest that it is possible: 1) to isolate highly enriched populations of DA neurons after sorting fluorescent midbrain cells derived from transgenic mice bearing a highly expressed 9.0 kb TH/LacZ construct and 2) to coax nascent neurons from the developing striatum or the human cell line (hNT) to adopt a DA phenotype. Using these three models and multidisciplinary analytical methods, which incorporate molecular, anatomical, biochemical and behavioral assessments, our specific goals for this proposal are: 1) establish the conditions which result in the most homogeneous yield of transgenic or hNT neurons exhibiting DA properties; 2) identify the culture conditions which are required for the survival, growth and preservation of a DA phenotype in transgenic or hNT cells; 3) establish the growth conditions that are required for the successful transplantation of transgenic, striatal or hNT cells in an animal model of Parkinson's disease. Knowledge gleaned from these studies will hopefully provide the building blocks needed to devise strategies for the treatment of Parkinson's and other diseases involving compromised DA systems.
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